Purpose In vitro fertilization with trophectoderm embryo biopsy and pre-implantation genetic screening with comprehensive chromosomal screening (PGS-CCS) for aneuploidy is becoming increasingly more popular. Embryos are cryopreserved and implanted in a subsequent frozen thawed embryo transfer cycle (FET). No studies have investigated differences in pregnancy outcomes by timing of trophectoderm biopsy relative to stages of blastocyst development. Methods Retrospective study of all patients ( n  = 363) at a single IVF center between January 1, 2013 and December 31, 2016 undergoing single embryo transfer with PGS-CCS where embryos were cryopreserved with subsequent FET. Embryo expansion and grading was assessed both at the time of biopsy and transfer. Pregnancy rates were analyzed by embryo expansion and embryo grading. Results Implantation, clinical pregnancy, and live birth rates improved significantly with increased embryo expansion at the time of embryo biopsy ( P  < 0.001). Pregnancy loss decreased with increases in embryo expansion prior to biopsy ( P  < 0.001). Superior live birth rates with PGS-CCS were seen when embryos were hatching at the time of biopsy ( p  < 0.001). For fresh and frozen embryo transfers without PGS-CCS, embryo expansion did not affect pregnancy outcomes. Conclusions PGS-CCS significantly increases implantation and live birth rates only if embryos are hatching at the time of biopsy. The embryo biopsy itself on a non-hatching embryo significantly damages the embryo in ways which are not reflected in future embryo expansion. IVF labs should wait until embryos hatch before performing trophectoderm biopsy.